The induction of lincomycin resistance in Lycopersicon peruvianum and Lycopersicon esculentum

Lincomycin-resistant calli were induced from both Lycopersicon esculentum and Lycopersicon peruvianum using N-mitroso-N-methylurea (NMU) mutagenesis. From these calli lincomycin-resistant plants were regenerated. For L. peruvianum it was shown that the resistant plants could be divided in two classes with respect to their resistance to lincomycin and its derivative clindamycin. The first class comprised plants which were resistant to 500 mg/l lincomycin and showed no shoot or root formation in the presence of clindamycin; the second class consisted of plants resistant to 2000 mg/l lincomycin and these plants were able to form shoots and roots on clindamycin containing media. Lincomycin is an inhibitor of peptidyltransferase; chloroplast encoded parts of this enzymatic function are sensitive for this antibiotic. Reciprocal crosses between our lincomycin resistant and wild type L. peruvianum plants indicated a maternal inheritance of the mutation.     

The biosynthesis of the lantibiotics epidermin,gallidermin, Pep5 and epilancin K7

Lantibiotics are antibiotic peptides that contain the rare thioether amino acids lanthionine and/or methyllanthionine. Epidermin, Pep5 and epilancin K7 are produced by Staphylococcus epidermidis whereas gallidermin (6L-epidermin) was isolated from the closely related species Staphylococcus gallinarum. The biosynthesis of all four lantibiotics proceeds from structural genes which code for prepeptides that are enzymatically modified to give the mature peptides. The genes involved in biosynthesis, processing, export etc. are found in gene clusters adjacent to the structural genes and code for transporters, immunity functions, regulatory proteins and the modification enzymes LanB, LanC and LanD, which catalyze the biosynthesis of the rare amino acids. LanB and LanC are responsible for the dehydration of the serine and threonine residues to give dehydroalanine and dehydrobutyrine and subsequent addition of cysteine SH-groups to the dehydro amino acids which results in the thioether rings. EpiD, the only LanD enzyme known so far, catalyzes the oxidative decarboxylation of the C-terminal cysteine of epidermin which gives the C-terminal S-aminovinylcysteine after addition of a dehydroalanine residue.Abbreviations Dha 2,3-didehydroalanine - Dhb 2,3-didehydrobutyrine - Lan lanthionine - Melan methyllanthionine     

Glycogenolysis during recovery from muscular work. The time course of phosphorylase activity is dependent on Pi concentration in the abdominal muscle of the shrimp Crangon crangon

1) Glycogen is degraded in the abdominal muscle of the shrimp Crangon crangon (Decapoda, Crustacea) during the recovery period following work. The regulation of post-exercise glycogen breakdown and the properties of glycogen phosphorylase (EC 2.4.1.1) have been studied: 2) Glycogen phosphorylase exists as unphosphorylated b-form and phosphorylated a-form, the latter contains 1 molecule phosphate/subunit. Both forms of phosphorylase are dimers, isoenzymes have not been detected. 3) The purified b-form is inactive in absence of AMP and has very low affinities for AMP and Pi. For half-maximum activation 0.33 +/- 0.04 mM AMP is necessary, and the Km-value for Pi at 1 mM AMP is 48 +/- 5 mM. IMP does not affect the activity of the b-form. 4) The a-form is active without effectors, its Km-value for Pi is 5.3 +/- 1.5 mM. The proportion of phosphorylase a increases in vivo, from about 25% at rest, to approximately 90% upon work and remains at this high level during the first minutes of recovery. 5) It is concluded that the glycogenolytic flux in the abdominal muscle of the shrimp even during post-exercise periods depends on the level of the a-form the activity of which is restricted in time and extent by the cytoplasmic Pi concentration (Kamp, G. & Juretschke, H. P. (1987) Biochim. Biophys. Acta 929, 121-127).     

Modification of the erythrocyte membrane by a non-specific lipid transfer protein

The non-specific phospholipid transfer protein purified from bovine liver has been used to modify the phospholipid content and phospholipid composition of the membrane of intact human erythrocytes. Apart from an exchange of phosphatidylcholine between the red cell and PC-containing vesicles, the protein appeared to facilitate net transfer of phosphatidylcholine from the donor vesicles to the erythrocyte and sphingomyelin transfer in the opposite direction. Phosphatidylcholine transfer was accompanied by an equivalent transfer (on a molar basis) of cholesterol. An increase in phosphatidylcholine content in the erythrocyte membrane from 90 to 282 nmol per 100 microliters packed cells was observed. Phospholipase C treatment of modified cells showed that all of the phosphatidylcholine which was transferred to the erythrocyte was incorporated in the lipid bilayer. The nonspecific lipid transfer protein used here appeared to be a suitable tool to modify lipid content and composition of the erythrocyte membrane, and possible applications of this approach are discussed.     

Biosynthesis and maturation of alpha-N-acetylglucosaminidase in normal and Sanfilippo B-fibroblasts

The biosynthesis of alpha-N-acetylglucosaminidase in normal and Sanfilippo B fibroblasts was studied by labeling cells with [35S]methionine and isolation of the enzyme by immunoprecipitation. The immunoprecipitated polypeptides were separated by polyacrylamide gel electrophoresis and visualized by fluorography. alpha-N-acetylglucosaminidase is synthesized as a precursor of an apparent mol. wt. of 87,000. Intracellular processing of the precursor yields two polypeptides of apparent mol. wts. of 73,000 and 76,000 via several intermediates. It is accomplished within 3 days after synthesis. Less than 30% of the newly synthesized precursor is secreted. In the presence of 10 mM NH4Cl, secretion is enhanced to more than 80%. In our study, no alpha-N-acetylglucosaminidase polypeptides could be detected in fibroblasts from patients affected with either the severe or mild form of Sanfilippo disease, type B.     

Recognition of different pools of phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii by phospholipase A2.

Phospholipase A2 (EC 3.1.1.4) from pig pancreas hydrolyzes phosphatidylglycerol in intact cells and isolated membranes of Acholeplasma laidlawii. Complete degradation of phosphatidylglycerol in intact cells at 37 degrees C does not result in lysis as shown by the retention of intracellular K+ ions and the cytoplasmic glucose-6-phosphatase, as well as the inability to detect activity of membrane-bound intracellular NADH-oxidase. A. laidlawii was grown on linoleic acid. Phospholipase A2 treatment of these cells at 5 degrees C, at which temperature the lipids are still in the liquid-crystalline state, results in a rapid breakdown of 50% of the phosphatidylglycerol. The residual phosphatidylglycerol can be hydrolyzed only at elevated temperatures and at much smaller rates, depending strongly on the incubation temperature. When membranes isolated from these cells are incubated at 5 degrees C, 70% of the phosphatidylglycerol is hydrolyzed immediately. The hydrolysis of the residual 30% is again strongly temperature dependent. Cells were grown on palmitate, elaidate, or oleate to investigate possible effects of the lipid phase transition on the accessibility of phosphatidylglycerol for phospholipase A2. Under conditions in which all the lipid is in the solid state, no hydrolysis occurs. When solid and liquid-crystalline lipid phases coexist, a limited hydrolysis of phosphatidylglycerol can be observed. The results demonstrate the disposition of phosphatidylglycerol in three different pools in the membrane of A. laidlawii. Phospholipase A2 has been used to discriminate between these pools and to estimate the amount of phosphatidylglycerol which is present in the liquid-crystalline phase. The present data, however, do not allow a definite localization of the phosphatidylglycerol pools.     

The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins.

An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.     

Lipid compartmentalization in erythrocytes parasitized by Plasmodium spp

Although reasonably well protected from the host immune system by the erythrocyte membrane, the intraerythrocytic malaria parasite has to make that membrane compatible with its own requirements for development and multiplication. The development of Plasmodium spp brings about major changes in the lipid composition of the host cell membrane, as well as in its physical properties. The parasite itself has a lipid composition that differs from that of the host cell and an intense lipid trafficking seems to occur between intracellular parasite and host cell membrane. Here, Ana Paula Sim?es, Ben Roelofsen and Jos Op den Kamp discuss how, despite serious methodological limitations and the existence of some conflicting results, an overall picture of lipid compartmentalization within the parasitized erythrocyte is perceived.